Use of L. reuteri for recovery of microbiota dysbiosis in early life

ABSTRACT

The invention concerns Lactobacillus reuteri for use in the prevention or treatment of microbiota dysbiosis, in particular, decreased levels of Actinobacteria and increased levels of Proteobacteria, in young mammals and in the prevention or treatment of disorders associated therewith. The microbiota dysbiosis may have been cause by numerous factors including being born by caesarean section, exposure to antibiotics in utero or after birth, or, parenteral feeding, hospitalizing, psychological stress or by gastrointestinal dysfunctions. The disorders that may be treated or prevented by preventing or treating microbiota dysbiosis include propensity to infection, allergy, type I diabetes mellitus, insulin resistance, type 2 diabetes, celiac disease, peripheral and central adiposity, obesity, necrotizing enterocolitis, inflammatory bowel disease, such as Crohn&#39;s disease and ulcerative colitis, and functional gastrointestinal disorders such as IBS, functional diarrhea, functional constipation, recurrent abdominal pain, and dyspepsia.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a National Stage of International ApplicationNo. PCT/EP2015/075164, filed on Oct. 29, 2015, which claims priority toEuropean Patent Application No. 14190941.6, filed on Oct. 29, 2014, theentire contents of which are being incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the field of infant health,in particular microbial colonization in the intestine of young mammals.The invention specifically relates to administration of L. reuteri toyoung mammals up to the age of three years in humans, and the equivalentage in animals, for promoting the recovery of microbiota dysbiosis. Theinvention also relates to the prevention or treatment of disordersassociated with microbiota dysbiosis.

BACKGROUND TO THE INVENTION

The present invention applies to all mammals, including animals andhumans.

Microbial colonisation of the infant intestine is a key triggeringfactor for the development of the infant gut and immune system.Immediately before birth, the gastro-intestinal tract of a baby isthought to be virtually sterile. During the normal process of birth, itencounters bacteria from the vagina, digestive tract and skin of themother and starts to become colonised. The fecal microbiota of ahealthy, vaginally-delivered, breast-fed infant of age 2 to 6 months isgenerally dominated by Bifidobacteria species with some Lactobacillusspecies and much lesser amounts of other bacteria groups such asBacteroides, and Proteobacteria, including the potentially pathogenicgenera such as Escherichia, Shigella and Klebsiella.

In the healthy, vaginally-delivered, breast-fed infant, Bifidobacteriaform the basis of the microbiota accounting for 30-50% of total bacteriain the infant gut.

This may be taken to represent the optimum microbiota for this agegroup, keeping in mind, however, that geographical factors influencelargely the microbiota. Thus, the microbiota of a healthyvaginally-delivered, breast-fed infant of age 2 to 6 months born in onepart of the world for example, may be different to that in another.

In any case, after the completion of weaning at about 2-3 years of age,a pattern of gut microbiota that resembles the adult pattern becomesestablished.

Microbiota dysbiosis may be defined as a significant deviation from abalanced microbiota, in terms of global microbiota profile, metabolismor levels of particular taxa. Microbiota dysbiosis is usually associatedwith and increased vulnerability to disease. For example, reduced levelsof Bifidobacterium are associated with increased risk of infection andother pathologies in infants.

A growing body of evidence indicates that the mode of delivery impactsconsiderably this colonization process. During vaginal delivery,microbiota from the mother's vagina and gut provides the bacteria thatinitiate intestinal colonization in the infant. By contrast, duringcaesarean section (C-section), there is no direct contact with themother fecal and vaginal microbiota, and the initial colonizers areessentially environmental bacteria. Consequently, C-section babies havesubstantially different founding microbiota composition and a delayedmicrobiota colonization compared to vaginally delivered babies.C-section microbiota is characterized by alterations in bacterialdiversity, reduced abundance of Bifidobacterium spp. and Bacteroidesspp. and increased levels of C. difficile [Adlerberth, I., (2009), AEWold Establishment of the gut microbiota in Western infants, ActaPædiatrica, 98, 229-238]. Microbiota alterations can be durable and havebeen detected several years after birth [Salminen, S., Gibson, G. R.,McCartney, A. L. and Isolauri, E. (2004), Influence of mode of deliveryon gut microbiota composition in seven year old children, Gut, 53,1388-1389]. Increased levels of the phylum Proteobacteria, includingpotentially pathogenic genera such as Escherichia, Shigella andKlebsiella, are also observed in infants born by C-section.

Caesarean delivery rates are increasing world-wide and currentlyrepresent more than 30% of births in very populated countries (e.g.U.S.A., China, Brazil).

Microbiota dysbiosis may be induced by events other than C-sectiondelivery including premature birth, exposure to antibiotics in utero,during delivery or after birth, parenteral feeding, hospitalization, orpsychological stress. Microbiota dysbiosis may also result fromgastrointestinal dysfunctions (digestive disorders, motility disorders,gastrointestinal reflux, slow gastrointestinal transit, oral feedingintolerance, constipation, diarrhea), Hirschsprung's disease, shortbowel syndrome, gastrointestinal infection and inflammation affectingthe gastrointestinal tract (such as Necrotisis enterocolitis) andobstruction pathologies.

Thus, young mammals, in particular, infants who have suffered any ofthese events are at risk of microbiota dysbiosis (aberrant microbiotacolonization).

It is believed that aberrant microbiota colonization can explain theincreased occurrence of certain disorders in those individuals born byC-section.

These disorders include severe and/or highly prevalent conditions suchas infection, allergy, type I diabetes mellitus [Neu, J., Rushing, J.(2011), Caesarean versus Vaginal Delivery: Long term infant outcomes andthe Hygiene Hypothesis, Clin. Perinatol., 38, 321-331], celiac disease,peripheral and central adiposity [Mesquita, (2013)] or obesity [HASGoldani, HAS, Bettiol H., Barbieri M A. et al. (2011), Caesareandelivery is associated with an increased risk of obesity in adulthood ina Brazilian birth cohort study, Am. J. Clin. Nutr., 93, 1344-7] thatsignificantly impair the quality of life of the individual and alsoresult in a considerably social and health care cost.

The risk of having these disorders is increased in those who havesuffered microbiota dysbiosis, whatever the cause of the dysbiosis.Thus, individuals who were born prematurely (either vaginally or byC-section), who were exposed to antibiotics in utero, during delivery orafter birth, or who were fed parenterally, who have suffered fromgastrointestinal dysfunctions (digestive disorders, motility disorders,gastrointestinal reflux, slow gastrointestinal transit, oral feedingintolerance), Hirschsprung's disease, inflammation affecting thegastrointestinal tract (such as Necrotizing enterocolitis) andobstruction pathologies in the three first years of life are also atrisk of having the above mentioned disorders.

On the other hand, as well as being a consequence of gastrointestinaldisorders, microbiota dysbiosis may actually cause them. Thus,microbiota dysbiosis may result in, for example, digestive disorders,motility disorders, gastrointestinal reflux, slow gastrointestinaltransit, oral feeding intolerance, Hirschsprung's disease, andinflammation affecting the gastrointestinal tract (such as Necrotizingenterocolitis) and obstruction pathologies.

Thus, preventing and treating microbiota dysbiosis occurring early inlife (for example, up to the age of three) may prevent or treat thenumerous disorders associated therewith, whether those disorders occurin infancy, or later in life.

The treatment of microbiota dysbiosis, includes re-establishing amicrobiota not significantly different from that observed healthy youngmammals, who are not experiencing microbiota dysbiosis. This means thatthe various bacterial populations are re-established in their optimalrelative abundance.

Pre- and probiotics have been proposed as a means to improve themicrobiota composition in C-section babies. However, up until now, thereare no published data demonstrating the capacity of probiotic tore-establish a healthy, balanced microbiota—one where the various gutbacteria are present in the optimal relative abundance.

The effect of the administration of L. reuteri to mothers with a familyhistory of atopic disease during the last 4 weeks of pregnancy and totheir babies from birth until 12 months has been described. While theprobiotic treatment resulted in a higher prevalence of L. reuteri in thestool samples from infants in the active as compared to the placebotreated group, during the first year of life, interestingly, theadministration of L. reuteri did not affect bifidobacteria or C.difficile colonization [Abrahamsson et al (2009) Probiotic Lactobacilliin Breast milk and infant stool in relation to oral intake during thefirst year of life, J. Ped. Gastroenterology and Nutrition 49, pp 1-6].

In international patent application WO2008116892, it is described howthe administration of Bifidobacterium lactis CNCM I-3446 to mice in ananimal model of C-section led to an increase in levels ofBifidobacterium breve, but had no effect on levels of Bifidobacteriumlongum.

In WO2010/010021 it was demonstrated that infants born by C-section havelower levels of fecal IgA compared to vaginally delivered infants.Administration of infant formula containing Bifidobacterium longumincreased levels of fecal IgA in the C-section infants. The microbiotaof the infants was not studied.

There is a need to prevent disorders associated with microbiotadysbiosis, including propensity to infection, allergy, type I diabetesmellitus, insulin resistance, type 2 diabetes, celiac disease,peripheral and central adiposity, obesity, necrotizing enterocolitis,inflammatory bowel disease, such as Crohn's disease and ulcerativecolitis, and functional gastrointestinal disorders such as IBS,functional diarrhea, functional constipation, recurrent abdominal painand dyspepsia.

SUMMARY OF THE INVENTION

The invention concerns Lactobacillus reuteri for use in the preventionor treatment of microbiota dysbiosis in a young mammal at risk of orsuffering from microbiota dysbiosis.

According to one aspect of the invention, the administration of L.reuteri increases or maintains levels of Actinobacteria and/or decreasesor maintains levels of Proteobacteria so that these levels are notsignificantly different from those in young mammals not suffering frommicrobiota dysbiosis.

The treatment generally for young mammals aged up to approximately threeyears of age in humans and the equivalent age in animals.

The invention also concerns the use of Lactobacillus reuteri forpreventing or treating disorders associated with said microbiotadysbiosis. The disorders may occur when the mammal is young, for exampleunder the age of three for a human and the equivalent age for an animal)or, they may occur later in life of the mammal.

The young mammal at risk of or suffering from microbiota dysbiosis mayhave been born or will be by caesarean section, been or is being exposedto antibiotics in utero, during delivery or after birth, or, been or isbeing fed parenterally. The young mammal at risk of or suffering frommicrobiota dysbiosis may be or may have been hospitalized, sufferingfrom psychological stress or from gastrointestinal dysfunctionsincluding digestive disorders, motility disorders, gastrointestinalreflux, slow gastrointestinal transit, oral feeding intolerance,constipation, diarrhea, Hirschsprung's disease, short bowel syndrome,gastrointestinal infection and inflammation affecting thegastrointestinal tract, such as Necrotizing enterocolitis, andobstruction pathologies.

The disorders that may be treated or prevented by preventing or treatingmicrobiota dysbiosis include propensity to infection, allergy, type Idiabetes mellitus, insulin resistance, type 2 diabetes, celiac disease,peripheral and central adiposity, obesity, necrotizing enterocolitis,inflammatory bowel disease, such as Crohn's disease and ulcerativecolitis, and functional gastrointestinal disorders such as IBS,functional diarrhea, functional constipation, recurrent abdominal pain,and dyspepsia.

The young mammal to be treated may be animal, or a human foetus,pre-term or term-born infant or a toddler.

The Lactobacillus reuteri may be administered to the foetus via theexpectant mother or may be administered to the young mammal directly orindirectly, via the lactating mother.

The administration period for the foetus is generally at least one week,preferably two weeks, more preferably at least one month and even morepreferably for the entirety of the gestation period. The administrationperiod for the young mammal is generally at least 4 weeks, preferably2-36 months in humans and the equivalent age in animals.

The Lactobacillus reuteri may be administered directly to the infant ortoddler in its pure form, or diluted in water or breast milk, in a foodsupplement, or in a milk fortifier, or in any milk support used duringtrophic feeding, or in a growing-up milk, or in a milk based drink or inan infant formula, such as a formula for premature infants, a starterformula or a follow-on formula, in a pharmaceutical or neutriceuticalcomposition, a growing-up milk, a milk-based drink, a food supplement,in a baby food, in an enteral nutritional product, a milk-based yoghurt,a dessert or pudding, in a biscuit, or cereal bar, cereal or, in afruit-based drink.

The Lactobacillus reuteri may be administered to the expectant orlactating mother orally, preferably in foods, drinks, dietarysupplements or pharmaceutical compositions.

The Lactobacillus reuteri may be administered to infant or toddler as adaily dose of from 1×10³ to 1×10¹², preferably, 1×10⁷ to 1×10¹¹ cfu(cfu=colony forming unit).

The Lactobacillus reuteri may be administered to the expectant orlactating mother, or infant as a composition comprising between 1×10³and 1×10¹² cfu/g of dry composition. Said composition may comprisefurther ingredients or prebiotics, preferably selected from inulin,fructo-oligosaccharide (FOS), short-chain fructo-oligosaccharide (shortchain FOS), galacto-oligosaccharide (GOS), fucosylated oligosaccharides,Sialylated oligosaccharides, human milk oligosaccharides (HMO) and cowmilk oligosaccharides (CMOS).

Said composition may comprise one or more additional probiotics,preferably selected from Bifidobacterium longum BB536 (ATCC BAA-999);Lactobacillus rhamnosus (CGMCC 1.3724), Bifidobacterium lactis (NCC2818)or mixtures thereof.

The Lactobacillus reuteri may be alive, inactivated or dead, fragmented,or in the form of fermentation products or metabolites, or a mixture ofany or all of these states.

Preferably, the Lactobacillus reuteri is Lactobacillus reuteri DSM17938.

The administration of the L. reuteri may be to a foetus via the mother.It may also be to a pre-term or term-born infant either directly or viamothers' milk.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Hierarchical clustering carried out on the mean weighted UniFracdistance matrix (i.e. UniFrac distances measuring the phylogeneticdissimilarity of the microbiota composition) of the 4 groups—Vaginal (V)and Caesarian (C) subjects fed Control (Ctrl) or L. reuteri formula (L.reuteri)—at the two ages—14 days (V2) and 4 month (V4). In thisdendogram, the groups are joined together in a hierarchical way from theclosest, that is the most phylogenetically similar, to the furthestapart, that is the most different

FIG. 2: Relative abundance of Phylum taxa in feces of each experimentalgroup at 2 weeks.

Control Vaginal (A), Control Cesarian (B), L. reuteri Vaginal (C), andL. reuteri Cesarian (D). Taxa that have a significantly differentrelative abundance are labeled in bold if more abundant compared toControl Vaginal group, or in bold and italic if less abundant than inControl Vaginal group.

FIG. 3: Relative abundance of Bifidobacterium spp. in feces of eachexperimental group at 2 weeks.

Control vaginal (Ctrl V), control cesarean (Ctrl C), L. reuteri vaginal(L. reuteri V) and L. reuteri cesarean (L. reuteri C). The error barindicates the SEmedian. P values for the pairwise comparisons betweeneach group vs. the control vaginal group (Ctrl V) are displayed(Wilcoxon test). Differences are considered significant at P<0.05.

DETAILED DESCRIPTION Definitions

In this specification, the following terms have the following meanings:

“Infants”: according to the Commission Directive 2006/141/EC of 22 Dec.2006 on infant formulae and follow-on formulae, article 1.2 (a), theterm “infants” means children under the age of 12 months.

“Pre-term infant” generally means an infant born before 37 weeksgestation.

“Term Born Infant” generally means an infant born after 37 weeksgestation.

“Toddler” generally means a child from when he can walk up to threeyears old.

“Young mammal” means in the context of the present invention a mammalwho has not entered puberty. This corresponds to infancy and childhoodin humans and the equivalent age in animals.

“Probiotic” means microbial cell preparations or components ormetabolites of microbial cells with a beneficial effect on the health orwell-being of the host [Salminen, S. et al. (1999); Probiotics: howshould they be defined, Trends Food Sci. Technol., 10 107-10]. Thedefinition of probiotic is generally admitted and in line with the WHOdefinition. The probiotic can comprise a unique strain ofmicro-organism, a mix of various strains and/or a mix of variousbacterial species and genera. In case of mixtures, the singular term“probiotic” can still be used to designate the probiotic mixture orpreparation. For the purpose of the present invention, micro-organismsof the genus Lactobacillus are considered as probiotics.

“Prebiotic” generally means a non-digestible food ingredient thatbeneficially affects the host by selectively stimulating the growthand/or activity of micro-organisms present in the gut of the host, andthus attempts to improve host health.

“Allergy” means an allergy which has been detected by a medical doctorand which can be treated occasionally or in a more durable manner. A“food allergy” is an allergy with respect to a food constituent.

“Infant formulae”: according to the Commission Directives 2006/141/EC of22 Dec. 2006 and/or 91/321/EEC of 14 May 1991 on infant formulae andfollow-on formulae, article 1.2 (c), the term “infant formulae” meansfoodstuffs intended for particular nutritional use by infants during thefirst four to six months of life and satisfying by themselves thenutritional requirements of this category of persons. It has to beunderstood that infants can be fed solely with infant formulas, or thatthe infant formula can be used by the carer as a complement of humanmilk. It is synonymous to the widely used expression “starter formula”.

“Follow-on formulae”: according to the Commission Directives 2006/141/ECof 22 Dec. 2006 and/or 91/321/EEC of 14 May 1991 on infant formulae andfollow-on formulae, article 1.2 (d), the term “follow-on formulae” meansfoodstuffs intended for particular nutritional use by infants aged overfour months and constituting the principal liquid element in aprogressively diversified diet of this category of persons.

“Growing-up milk”: milk-based nutritional composition especially adaptedto a child of between one year and three years old.

“Human Milk fortifier”: Nutritional composition for infants or youngchildren intended to be added to or diluted with human milk.

The term “hypoallergenic composition” means a composition which isunlikely to cause allergic reactions.

The term “sialylated oligosaccharide” means an oligosaccharide having asialic acid residue.

The term “fucosylated oligosaccharide” means an oligosaccharide having afucose residue.

All percentages are by weight unless otherwise stated.

As used in this specification, the words “comprises”, “comprising”, andsimilar words, are not to be interpreted in an exclusive or exhaustivesense. In other words, they are intended to mean “including, but notlimited to”.

Any reference to prior art documents in this specification is not to beconsidered an admission that such prior art is widely known or formspart of the common general knowledge in the field.

The inventors have demonstrated that administration of L. reuteri toyoung mammals, generally up to the age of three in humans and theequivalent age in mammals prevents microbiota dysbiosis.

The administration of L. reuteri thus prevents disorders associated withmicrobiota dysbiosis. These disorders include propensity to infection,allergy, type I diabetes mellitus, insulin resistance, type 2 diabetes,celiac disease, peripheral and central adiposity, obesity, necrotizingenterocolitis, inflammatory bowel disease, such as Crohn's disease andulcerative colitis, and functional gastrointestinal disorders such asIBS, functional diarrhea, functional constipation, recurrent abdominalpain, and dyspepsia.

Microbiota dysbiosis may be induced by C-section delivery, prematurebirth, exposure to antibiotics in utero or after birth, parenteralfeeding, hospitalization, or psychological stress, gastrointestinaldysfunctions (digestive disorders, motility disorders, gastrointestinalreflux, slow gastrointestinal transit, oral feeding intolerance,constipation, diarrhea), Hirschsprung's disease, short bowel syndrome,gastrointestinal infection and inflammation affecting thegastrointestinal tract (such as Necrotisis enterocolitis) andobstruction pathologies.

Thus, administration of L. reuteri will especially benefit individualswho are experiencing/have experienced at least one of these events.

According to a preferred embodiment of the invention, administration ofL. reuteri prevents or treats microbiota dysbiosis and disordersassociated therewith in infants who have been delivered by C-section.

According to further embodiments of the invention the L. reuteri isadministered to young mammal, who has been exposed to antibiotics inutero or after birth, or who has been fed parenterally for theprevention or treatment of microbiota dysbiosis and the disordersassociated therewith.

Young mammals, in particular infants who have suffered microbiotadysbiosis at birth, or in the months, or in the first three years afterbirth because of any of the events mentions previously may go on tomanifest any of the above mentioned disorders in the weeks or months oreven years after the microbiota dysbiosis first occurred. Thus,treatment with L. reuteri before birth (prenatally) or in the firstthree years of life in a human and the equivalent time in an animalprevents or treats disorders that:

-   -   (a) may occur in the first three years of life in humans (or the        equivalent age in animals) and    -   (b) may occur later in life (for example, up to the adulthood),        but which were initially caused by microbiota dysbiosis at birth        and/or in the three years after birth.

Examples of disorders that manifest themselves later in life are poorresistance to infection, allergy, asthma, atopic disease, type Idiabetes mellitus, insulin resistance, type 2 diabetes, celiac disease,peripheral and central adiposity, and obesity, necrotizingenterocolitis, and inflammatory bowel disorder/or disease.

While, generally, some of the causes of microbiota dysbiosis may occurat any time of life of an individual, the current invention focuses ontreatment of the young mammal up to the age of three years in humans,and the equivalent age for animals. The gut and the immune systemdevelops during this period and is modulated by the composition of themicrobiota colonizing the gut. Thus, preventing and treating microbiotadysbiosis during this critical time has a positive outcome forindividuals during and past early childhood and reaching into adulthood.

According to several embodiments of the invention, administration of L.reuteri to humans of up to three years of age, or to animals of theequivalent age, who are suffering from, or at risk of suffering frommicrobiota dysbiosis, prevents or treats disorders associated withmicrobiota dysbiosis.

According to one embodiment of the invention, the disorder associatedwith microbiota dysbiosis to be prevented or treated is poor resistanceto infection.

According to another embodiment of the invention, the disorderassociated with microbiota dysbiosis to be prevented or treated isperipheral and central adiposity, and/or obesity.

According to another embodiment of the invention, the disordersassociated with microbiota dysbiosis to be prevented or treated are typeI diabetes mellitus, insulin resistance, or type 2 diabetes.

According to another embodiment of the invention, the disordersassociated with microbiota dysbiosis to be prevented or treated areinflammatory bowel disease or necrotizing enterocolitis.

According to another embodiment of the invention, the disordersassociated with microbiota dysbiosis to be prevented or treated allergy,asthma, and/or atopic disease.

For all of the above mentioned embodiments, the microbiota dysbiosis,and thus, the ensuing disorder, may have been induced by any one or moreof C-section delivery, premature birth (vaginally or by C-section),exposure to antibiotics in utero or after birth, parenteral feeding inthe first three years of life, hospitalization, or psychological stress,gastrointestinal dysfunctions (digestive disorders, motility disorders,gastrointestinal reflux, slow gastrointestinal transit, oral feedingintolerance, constipation, diarrhea), Hirschsprung's disease, shortbowel syndrome, gastrointestinal infection and inflammation affectingthe gastrointestinal tract (such as Necrotisis enterocolitis) andobstruction pathologies.

According to a preferred embodiment of the invention, the administrationof L. reuteri is to infants and toddlers who have been delivered byC-section.

Any strain of L. reuteri may be used according to the invention.According to a preferred embodiment the L. reuteri is Lactobacillusreuteri DSM 17938, the L. reuteri strain owned by Biogaia AB, Sweden,having the scientific strain designation DSM 17938, formerly L. reuteriATCC 55730. The DSM identification refers to the DSMZ Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH Inhoffenstr. 7b, D-38124Braunschweig, Germany. DSM 17938. Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH Inhoffenstr. 7b D-38124 Braunschweig—Germany.

Other examples of L. reuteri suitable for use according to the inventionL. reuteri are ATCC PTA 6475, L. reuteri ATCC PTA 4659 and L. reuteriATCC PTA 5289 (available from Biogaia, Sweden), L. reuteri RC-14 (soldby Christian Hansen, France), L. reuteri NCIMB 30242 (sold as supplementcalled Cardioviva, by Micropharma Ltd., Canada) and L. reuteri DSMZ17648 (sold under the commercial name Pylopass, by Lonza, Switzerland)

The administration of the L. reuteri may be to a foetus, via the mother.It may also be to a pre-term or term-born infant, either directly or viamothers' milk.

The administration is generally up to the age of three years old, or theequivalent age in an animal.

Doses of Probiotic:

The probiotic may be administered as a daily dose and in the form of acomposition. The daily dose of L. reuteri administered to the expectantor breast feeding mother is from 1×10⁶ to 1×10¹² cfu, preferably 1×10⁸to 1×10¹¹ cfu (cfu=colony forming unit). The daily dose, suitable fornewborn babies, ranges from 1×10³ to 1×10¹², preferably, 1×10⁷ to 1×10¹¹cfu.

Thus, L. reuteri may be present in the composition in a wide range ofpercentages provided that it delivers the beneficial effect described.However, preferably, the L. reuteri is present in the composition in anamount equivalent to between 1×10³ and 1×10¹² cfu/g of dry composition.Preferably, for administration to the expectant or lactating mother orthe young adult, the probiotic is present in an amount equivalent tobetween 1×10⁴ to 1×10¹¹ cfu/g of dry composition. The amount ofprobiotic present per gram of dry composition for administration to theneonates, toddlers and children may be lower, preferably, 1×10⁶ to1×10⁹, and, of course, the daily doses described above should berespected.

The above doses include the possibilities that the bacteria are live,inactivated or dead, or even present as fragments such as DNA or cellwall or cytoplasmic materials, or as bacteria fermentation products oras bacteria metabolites. In other words, the quantity of bacteria whichthe formula contains is expressed in terms of the colony forming abilityof that quantity of bacteria as if all the bacteria were live,irrespective of whether they are, in fact, live, inactivated or dead,fragmented, or in the form of fermentation products or metabolites, or amixture of any or all of these states.

Method of Administration:

(i) Administration to Expectant Mothers:

The L. reuteri can be administered to the expectant mothers by variousways as long as it induces a contact with gastro-intestinal tract orvagina of the expectant mothers. For example, L. reuteri may beadministered vaginally as a capsule, suppository or tablet. Preferably,the administration is oral. Preferably, L. reuteri is orallyadministered in a composition as part of the food, drinks, tablets,capsules, pastilles or chewing gum, or dietary supplements of theexpectant mothers. The composition can also be administered in apharmaceutical composition. However, in pathological conditions or whenenteral feeding is otherwise used, the administration of the compositioncan be added to the enteral feeding composition. The enteral feeding maybe nasogastric, nasojejunal, or via a percutaneous endoscopicgastrostomy, or jejunostomy.

(ii) Administration to Newborn Mammals:

The L. reuteri can also be administered orally, directly to the youngmammal alone (pure or diluted in water or mother's milk for example) asa supplement (for example as a human milk fortifier supplement), or as apharmaceutical or nutraceutical composition, or as an ingredient in aninfant milk formula. Such a formula may be an infant “preterm formula”if the young mammal is born before term or has a low birth weight, a“starter formula” or a “follow-on formula”. A follow on formula isgenerally given to an infant of older than six months. The formula mayalso be an hypoallergenic (HA) formula in which the cow milk proteinsare hydrolysed. An example of a starter formula is given in Example 2.

(iv) Administration to Toddlers:

1. The L. reuteri can also be administered orally to toddlers and youngchildren in the form of in a pharmaceutical or neutriceuticalcomposition, a growing-up milk, a milk based drink, a food supplement,in a baby food, in an enteral nutritional product, a milk based yoghurt,a dessert or pudding, in a biscuit or cereal bar, cereal or in afruit-based drink.

(v) Administration to Animals:

The L. reuteri may also be administered orally to animals alone, or inwater, or in the form of a food supplement, a pharmaceutical ornutraceutical composition, or milk or pet food.

Administration with Other Compounds:

The L. reuteri can be administered alone (pure, or diluted in water ormilk, including breast milk, for example) or in a mixture with othercompounds (such as dietary supplements, nutritional supplements,medicines, carriers, flavours, digestible or non-digestibleingredients). Vitamins and minerals are examples of typical dietarysupplements. In a preferred embodiment, L. reuteri is administered in acomposition, for example, an infant formula, together with othercompounds that enhance the described beneficial effect on the youngmammals. Such synergistic compounds may be carriers or a matrix thatfacilitates the L. reuteri delivery to the intestinal tract or they mayotherwise enhance the effect of the composition on microbiota of theprogeny. Such compounds can be other active compounds thatsynergistically or separately influence the development of the entericnervous system in the infant and/or potentiates the effect of theprobiotic. An example of such synergistic compounds is maltodextrin. Oneof the effect of maltodextrin is to provide a carrier for the probiotic,enhancing its effect, and to prevent aggregation.

Other examples of synergistic compounds that may be included in thecompositions, especially infant formula, of the invention are prebioticcompounds. A prebiotic is a non-digestible food ingredient thatbeneficially affects the host by selectively stimulating the growthand/or activity of one or a limited number of bacteria in the colon, andthus improves host health. Such ingredients are non-digestible in thesense that they are not broken down and absorbed in the stomach or smallintestine and thus pass intact to the colon, where they are selectivelyfermented by the beneficial bacteria. Examples of prebiotics includecertain oligosaccharides, such as fructo-oligosaccharides (FOS), cowmilk oligosaccharides (CMOS) and galacto-oligosaccharides (GOS). Acombination of prebiotics may be used such as 90% GOS with 10% shortchain fructooligosaccharides such as the product sold under the trademark Raftilose® or 10% inulin such as the product sold under the trademark Raftiline®. Other examples of prebiotics that can be used in thecontext of the present invention include the group of oligosaccharidesobtained from milk or other sources, optionally containing sialic acid,fructose, fucose, galactose or mannose. Preferred prebiotics aresialo-oligosaccharides (SOS), fructo-oligosaccharides (FOS),galacto-oligosaccharides (GOS), isomalto-oligosaccharides (IMO),xylo-oligosaccharides (XOS), arabino-xylo oligosaccharides (AXOS),mannan oligosaccharides (MOS), oligosaccharides of soy, glycosylsucrose(GS), lactosucrose (LS), sialyl-lactose (SL), fucosyl-lactose (FL),lacto-N-neotetraose (LNNT), lacto-neotetraose (LNT), lactulose (LA),palatinose-oligosaccharides (PAO), malto-oligosaccharides, gums/orhydrolysates thereof, pectins, starches, and/or hydrolysates thereof. Aninfant formula according to the invention preferably further contains atleast one prebiotic in an amount of 0.3 to 10% of the total weight ofthe dry composition.

In particular, the human milk oligosaccharides, for example sialylatedoligosaccharides, described in WO 2012/069416 published on May 31, 2012may be included in the composition according to the invention. Thelatter oligosaccharides may act in synergy with the L. reuteri of theinvention to promote the recovery of C-section induced microbiotadysbiosis.

Other probiotics may be also administered. Preferably, the probiotic maybe selected for this purpose from the group consisting ofBifidobacterium, Lactobacillus, Lactococcus, Enterococcus,Streptococcus, Kluyveromyces, Saccharoymces, Candida, in particularselected from the group consisting of Bifidobacterium longum,Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve,Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillusacidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillussalivarius, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillusjohnsonii, Lactobacillus plantarum, Lactobacillus salivarius,Lactococcus lactis, Enterococcus faecium, Saccharomyces cerevisiae,Saccharomyces boulardii or mixtures thereof, preferably selected fromthe group consisting of Bifidobacterium longum NCC3001 (ATCC BAA-999),Bifidobacterium longum NCC2705 (CNCM 1-2618), Bifidobacterium longumNCC490 (CNCM 1-2170), Bifidobacterium lactis NCC2818 (CNCM I-3446),Bifidobacterium breve strain A, Lactobacillus paracasei NCC2461 (CNCMI-2116), Lactobacillus johnsonii NCC533 (CNCM 1-1225), Lactobacillusrhamnosus GG (ATCC53103), Lactobacillus rhamnosus NCC4007 (CGMCC1.3724), Enterococcus faecium SF 68 (NCC2768; NCIMB10415), and mixturesthereof.

The daily doses of carbohydrates, and all other compounds administeredwith the L. reuteri should always comply with the published safetyguidelines and regulatory requirements. This is particularly importantwith respect to the administration to new-born babies, especially thoseborn with low birth weight, very low or extremely low birth weight.

Infant formulas containing the L. reuteri may contain a protein sourcein an amount of not more than 4.0, 3.0 or 2.0 g/100 kcal, preferably 1.8to 2.0 g/100 kcal. The type of protein is not believed to be critical tothe present invention provided that the minimum requirements foressential amino acid content are met and satisfactory growth is ensuredalthough it is preferred that over 50% by weight of the protein sourceis whey. In one embodiment, the protein content is between 30% and 80%whey proteins. Thus, protein sources based on whey, casein and mixturesthereof may be used as well as protein sources based on soy. As far aswhey proteins are concerned, the protein source may be based on acidwhey or sweet whey or mixtures thereof and may include alpha-lactalbuminand beta-lactoglobulin in whatever proportions are desired.

The proteins may be intact or hydrolysed or a mixture of intact andhydrolysed proteins. It may be desirable to supply partially hydrolysedproteins (degree of hydrolysis between 2 and 20%), for example forinfants believed to be at risk of developing cows' milk allergy. Ifhydrolysed proteins are required, the hydrolysis process may be carriedout as desired and as is known in the art. For example, a whey proteinhydrolysate may be prepared by enzymatically hydrolysing the wheyfraction in one or more steps. If the whey fraction used as the startingmaterial is substantially lactose free, it is found that the proteinsuffers much less lysine blockage during the hydrolysis process. Thisenables the extent of lysine blockage to be reduced from about 15% byweight of total lysine to less than about 10% by weight of lysine; forexample about 7% by weight of lysine which greatly improves thenutritional quality of the protein source.

The composition may also comprise a source of carbohydrates and/or asource of fat. The infant formula may contain a source of lipids. Thelipid source may be any lipid or fat which is suitable for use in infantformulas. Preferred fat sources include palm olein, milk fat, high oleicsunflower oil and high oleic safflower oil. The essential fatty acids,linoleic and α-linolenic acid may also be added as small amounts of oilscontaining high quantities of preformed arachidonic acid anddocosahexaenoic acid such as fish oils or microbial oils. In total, thefat content is preferably such as to contribute between 30 to 55% of thetotal energy of the formula. The fat source preferably has a ratio ofn-6 to n-3 fatty acids of about 5:1 to about 15:1, for example about 8:1to about 10:1.

An additional source of carbohydrate may be added to the nutritionalcomposition. It preferably provides about 40% to about 80% of the energyof the nutritional composition. Any suitable carbohydrate may be used,for example sucrose, lactose, glucose, fructose, corn syrup solids,maltodextrin, or a mixture thereof.

Additional dietary fibre may also be added if desired. If added, itpreferably comprises up to about 5% of the energy of the nutritionalcomposition. The dietary fibre may be from any suitable origin,including for example soy, pea, oat, pectin, guar gum, acacia gum,fructo-oligosaccharide or a mixture thereof. Suitable vitamins andminerals may be included in the nutritional composition in an amount tomeet the appropriate guidelines.

Examples of minerals, vitamins and other nutrients optionally present inthe infant formula include vitamin A, vitamin B1, vitamin B2, vitaminB6, vitamin B 12, vitamin E, vitamin K, vitamin C, vitamin D, folicacid, inositol, niacin, biotin, pantothenic acid, choline, calcium,phosphorous, iodine, iron, magnesium, copper, zinc, manganese, chloride,potassium, sodium, selenium, chromium, molybdenum, taurine, andL-carnitine. Minerals are usually added in salt form. The presence andamounts of specific minerals and other vitamins will vary depending onthe intended infant population.

The infant formula may optionally contain other substances which mayhave a beneficial effect such as fibres, lactoferrin, nucleotides,nucleosides, and the like.

One or more essential long chain fatty acids (LC-PUFAs) may be includedin the composition. Examples of LC-PUFAs that may be added aredocosahexaenoic acid (DHA) and arachidonic acid (AA). The LC-PUFAs maybe added at concentrations so that they constitute greater than 0.01% ofthe fatty acids present in the composition.

One or more food grade emulsifiers may be included in the nutritionalcomposition if desired; for example diacetyl tartaric acid esters ofmono- and di-glycerides, lecithin and mono- or di-glycerides or amixture thereof. Similarly, suitable salts and/or stabilisers may beincluded. Flavours can be added to the composition.

Administration Period:

The duration of the administration may vary. While positive effects areexpected with relatively short duration of administration (for example,daily administration during one to two weeks for newborns), longerdurations are believed to provide an enhanced effect, or, at least, tomaintain the effect in older infants and toddlers (for example, aduration of three, five, eight, 12, 24 or 36 months). For administrationto animals, the corresponding durations apply.

The expectant mother may start to take the L. reuteri as soon as she isaware of her pregnancy. However, the administration period may alsostart before pregnancy starts, for example if the female is trying tobecome pregnant. Administration may start at any time after thepregnancy starts. It may start relatively late in the pregnancy,preferably at month 3, 4, 5, 6, 7, 8 or 9 of the pregnancy, in the caseof human pregnancy, or in corresponding periods for other mammals, or upto one week before the expected delivery date.

The period of administration can be continuous (for example, up to andincluding lactation up to weaning), or discontinuous. Continuousadministration is preferred for a more sustained effect. However, it isspeculated that a discontinuous pattern (for example, dailyadministration during one week per month, or during alternate weeks) caninduce positive effects on the progeny.

The administration may cover at least part of the gestation period andat least part of the lactation period if the newborn is fed withmother's milk, or the equivalent period, should the newborn not be fedwith mother's milk.

Preferably, the administration period to the expectant mother coverssubstantially the full length of the gestation period, although this maybe less. Similarly, the administration period for the lactating motherpreferably covers substantially the full length of the lactation period,although, again, this period may be less.

Preferably, the administration to the mother is by daily intake (to betaken once or twice a day), or weekly intake (to be taken one or twice aweek).

The L. reuteri may be administered to the infant directly. This is thecase particularly if the mother does not breastfeed, or after shediscontinues breastfeeding. However, an infant who is being breastfedmay also receive the L. reuteri by direct administration.

Preferably, the administration to the infant is by daily intake. Forexample, if the L. reuteri is administered as an infant formula, theadministration is with each feed, i.e. about four to about six timesdaily for infants less than one year old, the number of feeds reducingwith age.

The administration to the infant, either via breastfeeding, or by directadministration, or both methods, may be continued up until the age ofsix months or longer. Thus, the L. reuteri may be administered duringlactation, if lactation takes place, or after partial or full weaning.

Effect of the L. reuteri Administration:

The present inventors have surprisingly found that administration of L.reuteri to caesarian delivered infants leads, not simply to L. reutericolonization, as one might expect, but to the complete establishment ofa “normal” microbiota, characteristic of a vaginally born infant, whohas not been exposed to antibiotics or suffered events that are known todisturb the gut microbiota in infants. This has been demonstrated in aclinical study involving the genetic analysis of microbiota from stoolsamples of the infants (see Example 1).

From these data, it can be established that the administration of L.reuteri to young mammals, in particular to infants, treats or preventsmicrobiota dysbiosis. The microbiota dysbiosis in Example 1 was causedby C-section delivery. In general, microbiota dysbiosis may also becaused by premature birth (C-section or vaginally), exposure toantibiotics in utero or after birth, parenteral feeding,hospitalization, psychological stress, gastrointestinal dysfunctions(digestive disorders, motility disorders, gastrointestinal reflux, slowgastrointestinal transit, oral feeding intolerance, constipation,diarrhea), Hirschsprung's disease, short bowel syndrome,gastrointestinal infection and inflammation affecting thegastrointestinal tract (such as Necrotisis enterocolitis) andobstruction pathologies.

The administration of L. reuteri promotes the recovery of a “normal”microbiota population as that seen in infants born by vaginaldeliveries. The L. reuteri promotes the recovery of C-section induceddysbiosis. In the clinical experiment, detailed in Example 1, the effectof L. reuteri administration on the recovery of C-section inducedmicrobiota dysbiosis was evaluated. The inventors compared the evolutionof the microbiota composition in infants born by C-section and fedeither an infant formula containing L. reuteri DSM 17938 or a controlformula with similar composition but without the probiotic.

Delivery by C-section induced important changes in the global microbiotaprofile and taxa levels in the control group, especially 2 weeks afterbirth, but still detectable at 4 months of age.

FIG. 1 shows a dendogram clustering the four groups according to thephylogenetic similarity of their microbiota profile at both 2 weeks and4 months of age. At 2 weeks, the groups born via the vaginal routetightly cluster with the group born by C-section and fed the L reuteriformula, with a clear separation from the C-section group fed thecontrol formula. At this age the microbiota profile of infants born byC-section and fed the control formula was significantly different fromthose born by vaginal delivery (Cont C vs Cont V: p=0.009; Cont C vs L.reuteri V: p=0.01). In contrast, C-section infants fed with the L.reuteri formula had a microbiota profile closer to and not significantlydifferent from the vaginal delivery groups (L reuteri C vs Cont V:p=0.332; L. reuteri C vs L. reuteri V: p=0.682; L reuteri C vs Cont C:p=0.013).

The relative abundance at age 2 weeks of the Phylum taxa in thedifferent groups is shown in FIG. 2 and Table 1. The P values of thecomparisons between each group and the control vaginal group aredisplayed in Table 2.

TABLE 1 Median (SEmedian) values of the Phylum taxa relative abundancefor each group at 2 weeks of age¹ Ctrl V Ctrl C Lreuteri V Lreuteri CFirmicutes 49.0 (17.5) 59.5 (11.7) 60.3 (11.9) 58.5 (9.3) Actinobacteria44.5 (17.5) 0.5 (0.3) 36.0 (12.3) 25.4 (13.5) Proteobacteria 2.5 (1.0)27.6 (13.8) 2.4 (3.2) 7.3 (3.8) Bacteroidetes 0.1 (0.1) 0.0 (0.0) 0.0(0.0) 0.0 (0.0) Other 0.0 (0.0) 0.2 (0.1) 0.2 (0.2) 0.1 (0.0)Verrucomicrobia 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) ¹Values in boldand black indicate for a given group the Phyla that are significantlydifferent from the Control Vaginal group (cf. Table 2).

TABLE 2 P values for each pairwise comparison (Wilcoxon test) of thePhylum taxa median relative abundance at 2 weeks of age¹ Ctrl V vs. CtrlV vs. Ctrl V vs. Ctrl C Lreuteri V Lreuteri C Firmicutes 0.315 0.9050.314 Actinobacteria 0.008 0.720 0.426 Proteobacteria 0.011 0.842 0.809Bacteroidetes 0.224 0.628 0.021 Other 0.631 0.093 0.377 Verrucomicrobia**no P value derived since almost no infant has Verrucomicrobia¹Differences are considered significant at P < 0.05.

At two weeks, the relative abundance of Actinobacteria was stronglyreduced in infants born by C-section and fed the control formula,whereas the abundance of Proteobacteria was strongly increased in thisgroup. By contrast, the C-section group fed with the L. reuteri formulahad Actinobacteria and Proteobacteria levels close and not significantlydifferent from the vaginal delivery groups. Of note is the fact that theActinobacteria phylum contains beneficial micro-organisms such as thegenus Bifidobacterium, whereas the phylum Proteobacteria includespotentially pathogenic genera such as Escherichia, Shigella andKlebsiella.

Bifidobacterium was undetectable in most infants of the C-section,control group. This is illustrated in FIG. 3 which shows the relativeabundance of Bifidobacterium spp. in each group at 2 weeks of age.Bifidobacterium spp. was not detectable in most infants of the controlcaesarean group. As a consequence, the median value in this group was 0and significantly lower than the median value of the control group bornby vaginal delivery. By contrast, the levels of Bifidobacterium spp. inthe C-section group fed with the L. reuteri formula were close to andnot significantly different from the groups born by vaginal delivery.

The potentially harmful genera Klebsiella and Escherichia/Shigella werefar more abundant in the C-section, control group (3.0% and 18.2%,respectively) than in its vaginal-delivery counterpart (0.3% and 5.6%,respectively). Relative abundance of these genera was near vaginaldelivery levels in the C-section, L. reuteri group (1.2% and 7.4%,respectively).

At 4 months old, the global microbiota profile of each group vs. thecontrol vaginal group was no longer significant (FIG. 1), indicatingthat, as expected, the effect of C-section on microbiota composition wasmilder at the older age. Nevertheless, a significant effect of the modeof delivery on the relative abundance of several taxa was still observedin the group fed the control formula. C-section resulted in increasedrelative abundance of the phylum Firmicutes, of the familyEnterococcaceae and of the genera Enterococcus and Coprococcus, as wellas decreased relative abundance of the family Coriobacteriaceae and ofthe genus Collinsella.

Interestingly, compared to the vaginal delivery infants, no significantdifferences were observed in the relative abundance of any of the taxain the C-section group fed with L. reuteri supplemented formula.

Thus, L. reuteri containing formula induced a shift of the microbiota inC-section babies towards the vaginal delivery profile/levels, with astrong effect observed at two weeks and a complete microbiota recoveryat four months.

The L. reuteri formula promoted the recovery of the microbiota dysbiosisinduced by C-section. Specifically, L. reuteri induced an increase inActinobacteria levels, and a decrease in Proteobacteria levels so thatthe levels of these phyla were not significantly different fromvaginally born infants. This general recovery of microbiota dysbiosis inthe C-section infants towards a microbiota strongly resembling that ofvaginally-born infants represents significant advantage for maintainingthe health of those C-section infants, during infancy, childhood andlater in life. Thus, administration of L. reuteri may prevent or treatdisorders associated with C-section induced microbiota dysbiosis inyoung mammals. To the inventors knowledge, no oral treatment in theprior art has demonstrated this ability to treat microbiota dysbiosis.

The invention is beneficial to those young mammals that are suffering orare at risk of suffering microbiota dysbiosis. Specifically, populationsthat may benefit from administration of L. reuteri according to theinvention are those young mammals up to the age of 3 who:

-   -   have been delivered by cesarean section,    -   were exposed to antibiotics in utero during delivery or, once        born, were, or, are still being exposed to antibiotics,    -   were born prematurely (vaginally or by C-section),    -   were or are being fed parenterally,    -   experienced or are still experiencing hospitalization or        psychological stress,    -   suffered or are still suffering from gastrointestinal        dysfunctions including digestive disorders, motility disorders,        gastrointestinal reflux, slow gastrointestinal transit, oral        feeding intolerance, Hirschsprung's disease, and inflammation        affecting the gastrointestinal tract, such as Necrotisis        enterocolitis, and obstruction pathologies.

Although the invention has been described by way of example, it shouldbe appreciated that variations and modifications may be made withoutdeparting from the scope of the invention as defined in the claims.Furthermore, where known equivalents exist to specific features, suchequivalents are incorporated as if specifically referred in thisspecification.

Example 1

Clinical Study

Study Set-Up

A single-center, prospective, randomized, controlled, double-blindclinical trial of two groups in parallel was carried out. Healthy,full-term babies, anticipated to be exclusively formula-fed wereenrolled in the study. Infants were enrolled within the 48 hoursfollowing birth and were randomly assigned to one of 2 treatment groups:

1. Subjects receiving Starter Formula containing Lactobacillus reuteri(10⁸ cfu of L. reuteri per day) from birth to 6 months (n=60).

2. Subjects receiving Starter Formula without Lactobacillus reuteri frombirth to 6 months (n=60).

Stool samples were collected at 14 days and 4 months of infant age,refrigerated at 4° C. for a maximum of 10 hours after emission and keptfrozen at −80° C. until microbiota analysis was carried out.

Fecal Microbiota Analysis

Twenty infants in each group were selected to study the fecal microbiotacomposition. The selection was performed randomly, but with twostratification criteria: gender (10 males and 10 females in each group)and delivery type (10 infants born by vaginal and 10 infants born bycaesarean delivery in each group).

Fecal microbiota composition was measured at both time points bypyrosequencing of variable regions of the 16S RNA genes present in themicrobial population. DNA was extracted from fecal samples with theQiacube (QIAgen). Primers were designed as previously proposed [Hamady,M., Walker, J. J., Harris, J. K., N. et al. (2008), Error-correctingbarcoded primers for pyrosequencing hundreds of samples in multiplex,Nat. Methods, 5:235-237] to amplify the V1 to V3 variable regions of the16S gene, showing a high taxonomical informative level. Using barcodingtechniques, multiplex pyrosequencing was performed. Each sample wascharacterized by 1500 sequencing reads in average. High quality readswere identified and analyzed using QIIME analytical package [Caporaso,J. G., Kuczynski, J., Stombaugh, J. et al. (203.0), QIIME allowsanalysis of high-throughput community sequencing data, Nat Methods,7(5):335-6]. Reads were grouped into Operational Taxonomic Groups (OTUs)at 97% identity and further classified using the RDP-Classifier with 0.6confidence level [Wang, Q., Garrity, G. M., Tiedje, J. M. and Cole, J.R. (2007), Naive Bayesian classifier for rapid assignment of rRNAsequences into the new bacterial taxonomy, Appl. Environ. Microbiol.,73:5261-5267].

The statistical analysis of the microbiota data was performed by usingthe program R 2.14.1 [R Development Core Team. R: A language andenvironment for statistical computing. R Foundation for StatisticalComputing, Vienna, Austria. ISBN 3-900051-07-0, URLhttp://www.R-project.org/]. The effects of gender and delivery type weretested by using a NP MANOVA (Non-Parametric Multivariate ANalysis OfVariance) at each taxonomic level and 16S region with gender or deliverytype as one of the explanatory variables.

No significant effect of gender was observed and data from males andfemales were further analysed together. By contrast, the mode ofdelivery strongly modulated both the global microbiota profile and thetaxa relative abundance. To further explore the effect of the mode ofdelivery, data were analysed by comparing the following 4 groups:Vaginal delivery fed control (control vaginal) or L. reuteri (L. reuterivaginal) formula and C-section delivery fed control (control caesarean)or L. reuteri (L. reuteri cesarean) formula. NP MANOVA was used toanalyze the multivariate Euclidean distance data between two groups. TheWilcoxon signed-rank test was used for pairwise comparison of therelative abundance of each individual taxa.

Example 2

An example of the composition of an infant formula for use according tothe present invention is given below. This composition is given by wayof illustration only. The protein source is a mixture of 60% MSWP28 and40% casein.

Nutrient per 100 kcal per litre Energy (kcal) 100 670 Protein (g) 1.8312.3 Fat (g) 5.3 35.7 Linoleic acid (g) 0.79 5.3 α-Linolenic acid (mg)101 675 Lactose (g) 11.2 74.7 Prebiotic (100% GOS) (g) 0.64 4.3 Minerals(g) 0.37 2.5 Na (mg) 23 150 K (mg) 89 590 Cl (mg) 64 430 Ca (mg) 62 410P (mg) 31 210 Mg (mg) 7 50 Mn (μg) 8 50 Se (μg) 2 13 Vitamin A (μg RE)105 700 Vitamin D (μg) 1.5 10 Vitamin E (mg TE) 0.8 5.4 Vitamin K1 (μg)8 54 Vitamin C (mg) 10 67 Vitamin B1 (mg) 0.07 0.47 Vitamin B2 (mg) 0.151.0 Niacin (mg) 1 6.7 Vitamin B6 (mg) 0.075 0.50 Folic acid (μg) 9 60Pantothenic acid (mg) 0.45 3 Vitamin B12 (μg) 0.3 2 Biotin (μg) 2.2 15Choline (mg) 10 67 Fe (mg) 1.2 8 I (μg) 15 100 Cu (mg) 0.06 0.4 Zn (mg)0.75 5 L. reuteri 2 × 10⁷ cfu/g of powder DSM 17938

The invention claimed is:
 1. A method for treatment or reducing a riskof one or more disorders selected from the group consisting ofpropensity insulin resistance, type 2 diabetes, peripheral and centraladiposity, and obesity, wherein the one or more disorders are associatedwith microbiota dysbiosis occurring in a young human aged up to threeyears of age, the one or more disorders occurring in the young human orlater in the life of the young human, the method comprisingadministering a combination of Lactobacillus reuteri and one or moreadditional probiotics to the young human, wherein the young human issuffering from the microbiota dysbiosis and the administering iseffective to treat the microbiota dysbiosis.
 2. The method according toclaim 1, wherein the one or more additional probiotics comprise aBifidobacterium longum.
 3. The method according to claim 1, wherein theyoung human is selected from the group consisting of a human fetus, apre-term infant, a term-born infant and a toddler.
 4. The methodaccording to claim 1, wherein the young human is a fetus, and theLactobacillus reuteri is indirectly administered to the fetus viaadministration to the expectant mother.
 5. The method according to claim1, wherein the Lactobacillus reuteri is administered (i) directly to theyoung human or (ii) indirectly to the young human via administration tothe lactating mother.
 6. The method according to claim 1, wherein theLactobacillus reuteri is administered to the young human for a period ofat least one week.
 7. The method according to claim 1, wherein theLactobacillus reuteri is administered directly to the young human in atleast one form selected from the group consisting of a pure form,diluted in water or breast milk, in a food supplement, in a milkfortifier, in any milk support used during trophic feeding, in agrowing-up milk, in a milk based drink, in an infant formula, in apharmaceutical composition, in a nutriceutical composition, in a babyfood, in an enteral nutritional product, in a milk-based yoghurt, in adessert, in a pudding, in a biscuit, in a cereal bar, in a cereal and ina fruit-based drink.
 8. The method according to claim 1, wherein theyoung human is selected from the group consisting of a fetus with anexpectant mother and a breast-fed infant with a lactating mother, andthe administration to the young human is via oral administration to theexpectant mother or the lactating mother.
 9. The method according toclaim 1, wherein the young human is selected from the group consistingof an infant and a toddler, and the Lactobacillus reuteri isadministered to the infant or the toddler as a daily dose of 1×10³ to1×10¹² cfu (cfu=colony forming unit).
 10. The method according to claim1, wherein the young human is selected from the group consisting of afetus with an expectant mother, a breast-fed infant with a lactatingmother, and an infant, and the Lactobacillus reuteri is administered tothe expectant mother, the lactating mother, or the infant as acomposition comprising between 1×10³ and 1×10¹² cfu/g of drycomposition.
 11. The method according to claim 10, wherein thecomposition comprises further ingredients.
 12. The method according toclaim 1, wherein the one or more additional probiotics are selected fromthe group consisting of Bifidobacterium longum BB536 (ATCC BAA-999),Lactobacillus rhamnosus (CGMCC 1.3724), Bifidobacterium lactis (NCC2818)and mixtures thereof.
 13. The method according to claim 1, wherein theLactobacillus reuteri is Lactobacillus reuteri DSM
 17938. 14. The methodaccording to claim 1, wherein the Lactobacillus reuteri is administeredto the young human as a daily dose of 1×10³ to 1×10¹² cfu (cfu=colonyforming unit).
 15. The method according to claim 1, wherein the younghuman is selected from the group consisting of a fetus with an expectantmother, a breast-fed infant with a lactating mother, and an infant, andthe Lactobacillus reuteri is administered to the expectant mother, thelactating mother, or the infant as a composition comprising between1×10³ and 1×10¹² cfu/g of dry composition.